The understanding of many biological processes critically depends on the ability to track the complex dynamics of proteins and their interactions with other biomolecules in the context of a living cell. To date, single molecule methods have been effective in revealing the environmental and mechanistic heterogeneity of biological systems in vitro;however, observation of intracellular dynamics remains fundamentally limited due to the lack of suitable fluorescent labels. Even the best organic fluorophores available to date suffer from poor optical properties, low sustainable emission rates, and poor photostabilities, thus seriously limiting their application in single molecule studies. This problem becomes even more severe in the presence of the autofluorescent cellular background and the need to follow freely diffusing proteins within the cytosol or cellular compartments over extended time periods. To develop a new class of photostable single molecule probes, we propose to synthesize and optimize few-atom sized silver nanoclusters encapsulated into a specifically designed protective organic scaffold. Complementary to ssDNA-encapsulation and a parallel path toward this goal, these labels should exhibit emission rates that are 10-100-fold greater compared to the best organic dyes due to large oscillator strengths, excellent photostability, and nearly complete abolition of blinking pathways. Combined with the 10-fold background reduction in autofluorescence relative to blue excitation, the proposed fluorophores should exceed the 20-fold sensitivity enhancements needed for routine intracellular single molecule observation. The project is structured into two aims that detail 1) the scalable synthesis and photophysical characterization of the proposed few-atom sized clusters, and 2) their functionalization for biological targeting and incorporation into the modular tetrafunctional linker proposed in the parent grant for fluorogenic in vivo conjugation. Specifically designed organic ligands derived from published crystal structures of multinuclear silver clusters will serve not only as the templating scaffold for cluster formation, but also for attaching cross-linkable functional groups to rigidify the scaffold, thereby entrapping the cluster. The attachment of functional groups used in standard peptide chemistry will allow for further modification of the scaffold to enhance water solubility and bioconjugation. Based on ssDNA-encapsulated nanodot data, the proposed 3 atom sized silver clusters are likely to emit strongly in the red region, but the modular syntheses are readily extended to 2, 4, and 5 or possibly even larger cluster sizes to expand the available emitters, while tuning chemical and photophysical properties in a wide range of biologically relevant environments. The proposed small, highly emissive and photostable cluster-based emitters will be generally applicable to a wide range of intracellular imaging tasks, even in the presence of fast intracellular diffusion. Tying into the parent grant as a parallel path this competitive revision should greatly accelerate performing true intracellular single molecule studies detailing the nucleocytoplasmic trafficking of thioredoxins in response to oxidative stress. Public Health Relevance: Many diseases stem from improper protein and biomolecule interactions and the understanding of such interactions is critical to the improvement of disease diagnostics and treatment. While single molecule observations offer new opportunities to gain insights into the dynamics of protein interactions, these studies are currently limited due to the lack of bright fluorescent labels. New emitters based on few-atom silver clusters promise >10-fold improvements in photostability and sustainable emission rate - the two issues precluding intracellular single molecule dynamics from being followed. This competitive revision outlines the synthetic design of ligand-stabilized Ag nanodots to yield large quantities of highly stable, exceedingly bright next-generation biolabels. These generalized labels should then be available to the research community for deciphering signaling dynamics within living systems on bulk and single molecule levels.